It is known that an in vitro sensitivity test to antibiotics (antibiogram) provides, on the basis of current international guide lines, to set up a standardized bacterial suspension to test against optimized concentrations (breakpoints) or scale dilutions of antibiotics (M.I.C).
The number of bacteria analyzed must be standardized irrespective of the sensitivity of the method adopted for the test.
The preparation of an inoculum is one of the most critical passages in every sensitivity test or antibiogram. The inoculum can significantly influence the dimensions of the inhibition area.
The choice of the inoculum method is mainly conditioned by practical considerations, but the results are better if some form of standardization is adopted, such as the comparison of the density of the suspension of the micro-organisms to a determinate standard of turbidity or to an equivalent latex, or by making photometric measurements.
In particular, the standardization method most widely used for the standardization of the inoculum uses the McFarland turbidity standard, typically used in microbiology as a reference to regulate the turbidity of bacterial suspensions so as to have the number of bacteria within a certain range.
It is known that, in classical microbiology, McFarland turbidity is the usual one to be able to begin tests on antibiotics (antibiogram) or to perform a phenotypical identification. Normally, the growth in a Petrie dish shows the isolated colonies and these bacteria are diluted until the McFarland value of 0.5 is obtained, that is, the level of turbidity.
The McFarland standards (0.1, . . . , 0.3, . . . , 0.5, . . . , 1, . . . , 2, . . . , 3, . . . , 4, . . . ) can be prepared by means of the addition of specific volumes of sulfuric acid and barium chloride dehydrate to obtain a solution of barium sulfate with a specific optical density.
The most common standard used is the McFarland 0.5 standard which supplies a visually comparable standard with that of a bacterial suspension, in a sterile saline solution or growth broth, containing approximately 1.5*108CFU/ml.
Once standardized, the suspension of the inoculum should be used within 15 minutes of the preparation.
The application UD2009A000048 in the name of the Applicant proposes a method to rapidly obtain a standardization of McFarland turbidity, to be combined with the execution of a direct antibiogram. This method is particularly useful for patients affected by the presence of bacteria in biological liquids and in urine in particular, and to begin the antibiotic therapy quickly.
The application US-A-2005/254055 is also known which describes a method to monitor the cellular growth and the concentration in a dynamic environment of cell cultures.
As an alternative bacterial identification technique it is also known, for example from the international application WO-A-2009/065580 (WO'580), to carry out the identification of pathogens, such as viruses, bacteria or other micro-organisms, by means of mass spectrophotometric measurements of their protein profile, obtained from pathogens directly precipitated as pellets from biological fluid samples centrifuged after culture growth in a defined solid growth medium, for example a solid medium in a Petrie dish. The growth of bacteria in a Petrie dish normally occurs in a time which varies from six to twenty hours. In such culture media even contaminating bacteria can grow, and in any case the identification method, in order to be applied to the mass spectrophotometer, must bear in mind that:                the mass of bacteria of the pellet is put into contact with a conventional matrix to ionize the sample and must come within a range of Daltons which varies from 3,000 to 15,000 Daltons, with a Dalton mass minimum equal to 2,500. Values above this range risk blocking the mass spectrophotometer.        
It must also be noted that the pellet obtained from the positive sample with the method described in WO'580 does not allow to select the pollutants from the bacteria responsible for the infection in progress.
Consequently, WO'580 has its limitations in discriminating a maximum of two bacteria in the sample to be analyzed, in that the detection algorithm to identify the bacteria using an identification library is not able to discriminate if the number of bacteria present in a sample is more than the two species combined. Consequently the known technique suffers in particular from the presence of other contaminating bacteria, as well as the bacterium responsible for the infection.
Moreover, this technology can often give mass spectrums which are not very clear with problems of basic interference. Furthermore, the centrifugation method contributes to lengthening the times and increases the overall costs of analysis.
Purpose of the present invention is to perfect a method and achieve an apparatus for diagnostic analyses, in particular to identify pathogens, such as bacteria, viruses or other micro-organisms, which is both quick, economical, reliable, even in the case of the presence of other contaminating pathogens as well as the pathogens responsible for the infection, and which simplifies a subsequent identification step by means of mass spectrophotometry.
The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages.